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IrSPI, a tick serine protease inhibitor involved in tick feeding and Bartonella henselae infection.

Liu XY, de la Fuente J, Cote M, Galindo RC, Moutailler S, Vayssier-Taussat M, Bonnet SI - PLoS Negl Trop Dis (2014)

Bottom Line: IrSPI silencing impaired tick feeding, as well as resulted in reduced bacterial load in tick SGs.This study provides a comprehensive analysis of I. ricinus SG transcriptome and contributes significant genomic information about this important disease vector.This in-depth knowledge will enable a better understanding of the molecular interactions between ticks and tick-borne pathogens, and identifies IrSPI, a candidate to study now in detail to estimate its potentialities as vaccine against the ticks and the pathogens they transmit.

View Article: PubMed Central - PubMed

Affiliation: USC INRA Bartonella-Tiques, French National Institute of Agricultural Research (UMR BIPAR ENVA-ANSES-UPEC), Maisons-Alfort, France.

ABSTRACT
Ixodes ricinus is the most widespread and abundant tick in Europe, frequently bites humans, and is the vector of several pathogens including those responsible for Lyme disease, Tick-Borne Encephalitis, anaplasmosis, babesiosis and bartonellosis. These tick-borne pathogens are transmitted to vertebrate hosts via tick saliva during blood feeding, and tick salivary gland (SG) factors are likely implicated in transmission. In order to identify such tick factors, we characterized the transcriptome of female I. ricinus SGs using next generation sequencing techniques, and compared transcriptomes between Bartonella henselae-infected and non-infected ticks. High-throughput sequencing of I. ricinus SG transcriptomes led to the generation of 24,539 isotigs. Among them, 829 and 517 transcripts were either significantly up- or down-regulated respectively, in response to bacterial infection. Searches based on sequence identity showed that among the differentially expressed transcripts, 161 transcripts corresponded to nine groups of previously annotated tick SG gene families, while the others corresponded to genes of unknown function. Expression patterns of five selected genes belonging to the BPTI/Kunitz family of serine protease inhibitors, the tick salivary peptide group 1 protein, the salp15 super-family, and the arthropod defensin family, were validated by qRT-PCR. IrSPI, a member of the BPTI/Kunitz family of serine protease inhibitors, showed the highest up-regulation in SGs in response to Bartonella infection. IrSPI silencing impaired tick feeding, as well as resulted in reduced bacterial load in tick SGs. This study provides a comprehensive analysis of I. ricinus SG transcriptome and contributes significant genomic information about this important disease vector. This in-depth knowledge will enable a better understanding of the molecular interactions between ticks and tick-borne pathogens, and identifies IrSPI, a candidate to study now in detail to estimate its potentialities as vaccine against the ticks and the pathogens they transmit.

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Influence of IrSPI silencing on tick feeding and tick SG infection by B. henselae.IrSPI-siRNA (siRNA) or nuclease free water (control) were microinjected into the body of B. henselae-infected I. ricinus females before ticks were fed a non-infected blood meal during 7 days. A) Quantitative RT-PCR analysis of IrSPI gene expression levels in pools of eight tick SGs from IrSPI-siRNA injected ticks and controls. The results are represented as the mean ± SEM of qRT-PCR performed in triplicate. B) Weight evaluation of IrSPI-siRNA injected tick body mass compared to controls. The results are represented as the mean ± SEM of eight ticks weighed individually. C) Quantitative PCR analysis of bacterial load in pools of eight tick SGs from IrSPI-siRNA injected ticks and controls. The results are represented as the mean ± SEM of qPCR performed in triplicate.
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pntd-0002993-g005: Influence of IrSPI silencing on tick feeding and tick SG infection by B. henselae.IrSPI-siRNA (siRNA) or nuclease free water (control) were microinjected into the body of B. henselae-infected I. ricinus females before ticks were fed a non-infected blood meal during 7 days. A) Quantitative RT-PCR analysis of IrSPI gene expression levels in pools of eight tick SGs from IrSPI-siRNA injected ticks and controls. The results are represented as the mean ± SEM of qRT-PCR performed in triplicate. B) Weight evaluation of IrSPI-siRNA injected tick body mass compared to controls. The results are represented as the mean ± SEM of eight ticks weighed individually. C) Quantitative PCR analysis of bacterial load in pools of eight tick SGs from IrSPI-siRNA injected ticks and controls. The results are represented as the mean ± SEM of qPCR performed in triplicate.

Mentions: The transcript encoding a BPTI/Kunitz type serine protease inhibitor, thereafter named IrSPI (for I. ricinus serine protease inhibitor; GenBank accession number: KF531922), that was overexpressed in response to bacteril infection was selected for functional analysis in ticks. RNAi was used to evaluate the effect of IrSPI silencing on tick feeding as well as tick salivary gland infection by B. henselae. siRNA injection was performed in females arising from infected nymphs and larvae, prior to the provision of an additional uninfected blood meal, to ensure “total” SG infection of the adult females (see material and methods section). IrSPI transcript abundance was suppressed by 90% (p = 0.001) in ticks that received IrSPI-siRNA compared to ticks that only received control injections (Figure 5A). The mean weight of siRNA-injected females was significantly decreased by 1.6-fold (12.7 mg±1.7 vs. 20.3 mg±2.1), when compared to controls (Figure 5B). B. henselae load within SGs was significantly reduced by 2.5-fold in IrSPI-siRNA-injected ticks when compared to mock injected ticks (1.6×10−4±0.1 and 3.9×10−4±0.2 per actin gene copy, respectively) (Figure 5C).


IrSPI, a tick serine protease inhibitor involved in tick feeding and Bartonella henselae infection.

Liu XY, de la Fuente J, Cote M, Galindo RC, Moutailler S, Vayssier-Taussat M, Bonnet SI - PLoS Negl Trop Dis (2014)

Influence of IrSPI silencing on tick feeding and tick SG infection by B. henselae.IrSPI-siRNA (siRNA) or nuclease free water (control) were microinjected into the body of B. henselae-infected I. ricinus females before ticks were fed a non-infected blood meal during 7 days. A) Quantitative RT-PCR analysis of IrSPI gene expression levels in pools of eight tick SGs from IrSPI-siRNA injected ticks and controls. The results are represented as the mean ± SEM of qRT-PCR performed in triplicate. B) Weight evaluation of IrSPI-siRNA injected tick body mass compared to controls. The results are represented as the mean ± SEM of eight ticks weighed individually. C) Quantitative PCR analysis of bacterial load in pools of eight tick SGs from IrSPI-siRNA injected ticks and controls. The results are represented as the mean ± SEM of qPCR performed in triplicate.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4109860&req=5

pntd-0002993-g005: Influence of IrSPI silencing on tick feeding and tick SG infection by B. henselae.IrSPI-siRNA (siRNA) or nuclease free water (control) were microinjected into the body of B. henselae-infected I. ricinus females before ticks were fed a non-infected blood meal during 7 days. A) Quantitative RT-PCR analysis of IrSPI gene expression levels in pools of eight tick SGs from IrSPI-siRNA injected ticks and controls. The results are represented as the mean ± SEM of qRT-PCR performed in triplicate. B) Weight evaluation of IrSPI-siRNA injected tick body mass compared to controls. The results are represented as the mean ± SEM of eight ticks weighed individually. C) Quantitative PCR analysis of bacterial load in pools of eight tick SGs from IrSPI-siRNA injected ticks and controls. The results are represented as the mean ± SEM of qPCR performed in triplicate.
Mentions: The transcript encoding a BPTI/Kunitz type serine protease inhibitor, thereafter named IrSPI (for I. ricinus serine protease inhibitor; GenBank accession number: KF531922), that was overexpressed in response to bacteril infection was selected for functional analysis in ticks. RNAi was used to evaluate the effect of IrSPI silencing on tick feeding as well as tick salivary gland infection by B. henselae. siRNA injection was performed in females arising from infected nymphs and larvae, prior to the provision of an additional uninfected blood meal, to ensure “total” SG infection of the adult females (see material and methods section). IrSPI transcript abundance was suppressed by 90% (p = 0.001) in ticks that received IrSPI-siRNA compared to ticks that only received control injections (Figure 5A). The mean weight of siRNA-injected females was significantly decreased by 1.6-fold (12.7 mg±1.7 vs. 20.3 mg±2.1), when compared to controls (Figure 5B). B. henselae load within SGs was significantly reduced by 2.5-fold in IrSPI-siRNA-injected ticks when compared to mock injected ticks (1.6×10−4±0.1 and 3.9×10−4±0.2 per actin gene copy, respectively) (Figure 5C).

Bottom Line: IrSPI silencing impaired tick feeding, as well as resulted in reduced bacterial load in tick SGs.This study provides a comprehensive analysis of I. ricinus SG transcriptome and contributes significant genomic information about this important disease vector.This in-depth knowledge will enable a better understanding of the molecular interactions between ticks and tick-borne pathogens, and identifies IrSPI, a candidate to study now in detail to estimate its potentialities as vaccine against the ticks and the pathogens they transmit.

View Article: PubMed Central - PubMed

Affiliation: USC INRA Bartonella-Tiques, French National Institute of Agricultural Research (UMR BIPAR ENVA-ANSES-UPEC), Maisons-Alfort, France.

ABSTRACT
Ixodes ricinus is the most widespread and abundant tick in Europe, frequently bites humans, and is the vector of several pathogens including those responsible for Lyme disease, Tick-Borne Encephalitis, anaplasmosis, babesiosis and bartonellosis. These tick-borne pathogens are transmitted to vertebrate hosts via tick saliva during blood feeding, and tick salivary gland (SG) factors are likely implicated in transmission. In order to identify such tick factors, we characterized the transcriptome of female I. ricinus SGs using next generation sequencing techniques, and compared transcriptomes between Bartonella henselae-infected and non-infected ticks. High-throughput sequencing of I. ricinus SG transcriptomes led to the generation of 24,539 isotigs. Among them, 829 and 517 transcripts were either significantly up- or down-regulated respectively, in response to bacterial infection. Searches based on sequence identity showed that among the differentially expressed transcripts, 161 transcripts corresponded to nine groups of previously annotated tick SG gene families, while the others corresponded to genes of unknown function. Expression patterns of five selected genes belonging to the BPTI/Kunitz family of serine protease inhibitors, the tick salivary peptide group 1 protein, the salp15 super-family, and the arthropod defensin family, were validated by qRT-PCR. IrSPI, a member of the BPTI/Kunitz family of serine protease inhibitors, showed the highest up-regulation in SGs in response to Bartonella infection. IrSPI silencing impaired tick feeding, as well as resulted in reduced bacterial load in tick SGs. This study provides a comprehensive analysis of I. ricinus SG transcriptome and contributes significant genomic information about this important disease vector. This in-depth knowledge will enable a better understanding of the molecular interactions between ticks and tick-borne pathogens, and identifies IrSPI, a candidate to study now in detail to estimate its potentialities as vaccine against the ticks and the pathogens they transmit.

Show MeSH
Related in: MedlinePlus